Arguably the most significant advance in the field of immunology in the last decade is delineation of the pivotal role of regulatory T cells (Tregs) in the maintenance of self-tolerance. While Tregs are just now entering early phase clinical trials, data gleaned from basic and translational studies to-date suggest enormous potential to intervene in human disease. Our work is founded on the premise that optimal clinical application of Tregs will require comprehensive understanding of both the mechanism of Treg action in vivo, as well as the factors by which Tregs are themselves regulated;exploring these issues is the focus of the proposal. During the prior funding period we made the unexpected observation that the function of regulatory T cells was impaired in the setting of an acute transplant when compared to a chronic graft that has [unreadable]healed in[unreadable] for a month. This observation led us to hypothesize that innate activation resulting from the transplant procedure with attendant trauma, ischemia, and bacterial contamination, inactivates Tregs via innate signals resulting in dendritic cell activation and maturation[unreadable]we refer to this process of functional Treg silencing as counterregulation. The notion that Treg function is modulated by innate signals is now well-documented. In the transplant arena, the issue is less well-studied but has potentially broad import being relevant not only in considering therapeutic Treg delivery but also in that it may explain why tolerogenic strategies are successful in pathogen-free mice, but fail in primates and in humans who experience perpetual innate activation. The current proposal will exploit findings in the preceding funding period to further our understanding of the mechanism of action of Tregs in vivo. Aim I will explore the contribution of APCs in promoting regulation and will assess whether Th lineage plasticity, specifically the conversion of naive graft-reactive T cells to Foxp3+ Tregs, contributes to regulation in general and linked suppression in particular. Aim II examines the in vivo mechanism of counter-regulation exploiting the model of acute and chronic graft transplantation and differences in their ability to be regulated. We define contributions of direct and indirect presentation pathways, and delineate the factors that mediate counter-regulation of Tregs. We also probe further our finding that a hallmark of counter-regulation in vivo is reduction in Treg Foxp3 expression. We assess whether Foxp3 down-regulation signifies lineage conversion to a Th1 or Th17 phenotype (or a polyfunctional lineage), and if so, whether these alterations are permanent or revert to the basal Treg phenotype following resolution of innate danger signals. Finally, Aim III assesses a clinically relevant implication of our general thesis, whether systemic infection induced innate activation leads to counter regulatory signals subverting Treg function and prevention of Treg induced graft acceptance. Collectively, the work is directed toward clinical application of Tregs and is highly in vivo biased thereby adding to the translational relevance of the findings that will be generated.